Based on a common folding pattern, proteins may be assigned to fewer than 1000 protein families (Chothia, Nature, 357:543-544 (1992) and Gonnet et al., Science, 256:1443-1445 (1992). Despite the conservation of overall structure, these families of proteins are often characterized by a large repertoire of specificities, as demonstrated, for example, by the immunoglobulin superfamily. Protein-modules are the building blocks which are conserved within a protein family. Among the receptors for various growth and differentiation regulating hormones, structural predictive algorithms identified a common protein motif, now referred to as the cytokine receptor homology domain as described by Bazan, Proc. Natl. Acad. Sci. USA, 87:6934-6938 (1990) and Thoreau et al., FEBS Lett., 282:26-31 (1991). This domain consists of a pair of structurally similar modules which were proposed to adopt a seven .beta.-strand fold with similarity (Bazan, supra and Patthy, Cell, 61:13-14 (1990) to the domain 2 of CD4 [Wang et al., Nature, 348:411-418 (1990) and Ryu et al., Nature, 348:419-426 (1990)] and to the fibronectin type III repeats [Main et al., Cell, 71:671-678 (1992)]. Experimental support for the proposed structure of the cytokine receptor homology domain was provided by the crystallographic solution of the three dimensional structure of the growth hormone receptor (GHR), a member of this receptor family as described by De Vos et al., Science, 255:306-312 (1992).
Tissue factor (TF) has been predicted to be a distant member of the cytokine receptor family, with structural similarity closer to the interferon receptors [Bazan, supra, and Edgington et al., Thrombosis and Haemostasis, 66:67-79 (1991)] than to the receptors for growth hormone, erythropoietin, colony stimulating factors or several of the interleukins. TF initiates the blood coagulation cascades [Davie et al., Biochem., 30:10363-10370 (1991)] by binding of its ligand, the serine protease Factor VIIa (VIIa) resulting in the formation of a catalytically active enzyme-cofactor complex as described by Edgington et al., supra. In addition, TF serves as a mediator for the auto-activation of VII to VIIa as described by Nakagaki et al., Biochem., 30:10819-10824 (1991).
Upon assembly with TF, VIIa exhibits enhanced catalytic activity evidenced by hydrolysis of small peptidyl [Ruf et al., J. Biol. Chem., 266: 2158-2166, (1991); and Lawson et al., J. Biol. Chem., 267: 4834-4843, (1992)] as well as protein substrates [Ruf et al., J. Biol. Chem., 266: 2158-2166, (1991); and Silverberg et al., J. Biol. Chem., 252: 8481-8488, (1977); Osterud et al., Proc. Natl. Acad. Sci., USA, 74: 5260-5264, (1977); Bom et al., Biochem J., 265: 327-336, (1990); and Lawson et al., J. Biol. Chem., 266: 11317-11327, (1991)]. Cleavage of small peptidyl substrates is efficient with micromolar concentrations of the divalent cation, calcium, Ca.sup.2+, whereas, the activation of macromolecular substrates such as the zymogen Factor X (X) require the presence of Ca.sup.2+ at concentrations consistent with saturation of the gamma-carboxylated amino-terminal domain of VIIa [Ruf et al., J. Biol. Chem., 266: 15719-15725, (1991)].
By serving as a cell surface receptor for a protease, TF does not follow the functional paradigm of this receptor family which is characterized by binding of growth and differentiation regulating hormones. Nevertheless, ligand binding by TF is mediated entirely by the structurally conserved extracellular domain [Ruf et al., J. Biol. Chem., 266:2158-2166 (1991) and Ruf et al., J. Biol. Chem., 266:15719-15725 (1991)], as observed for other cytokine receptors as described by De Vos, supra, and Fukunaga et al., EMBO J., 10:2855-2865 (1991).
The assembly of VIIa with its receptor TF results in the formation of a proteolytically competent enzyme-cofactor complex. In order to define the structure which mediates TF function, a combination of antibody, chemical cross-linking and mutational analysis has been applied. See, Ruf et al., Biochem. J., 278:729-733 (1991); Rehemtulla et al., J. Biol. Chem., 266:10294-10299 (1991); Rehemtulla et al., Biochem. J., 282:737-740 (1992); Ruf et al., Proc. Natl. Acad. Sci. USA, 88:8430-8434 (1991); Ruf et al., J. Biol. Chem., 267:6375-6381 (1992); and Ruf et al., J. Biol. Chem., 267:22206-22210 (1992).
As described in the latter reference, a discrete functional region in the carboxyl (C) module of the TF extracellular domain has been extensively characterized by scanning alanine mutagenesis. The detailed analysis of these mutants in the amino acid residue sequence from positions 157-168 demonstrated that residues contributing to function were neither required for high affinity binding of VIIa nor for the enhanced catalytic efficiency of the bound VIIa to hydrolyze small peptidyl substrates. Rather, residues in this particular region specifically contributed to efficient cleavage of the macromolecular substrate Factor X, indicating the existence of functionally important substrate-cofactor interactions.
Based on comparison with GHR and cytokine homology domain, previous studies have implicated the structural modules of TF as the region required for VIIa ligand binding. Sequence-specific antibodies raised against peptides corresponding to residues 40-71 of TF (C.sub.N -C'.sub.N - and E.sub.N -F.sub.N -loops) inhibited binding of VIIa to TF as described by Ruf et al., Biochem. J., 278:729-733 (1991). Monoclonal antibodies that completely blocked binding of VIIa have further been assigned to an epitope in the carboxyl aspect of the residue 1-83 sequence. Spatial proximity of residues 44-84 to ligand was further demonstrated by chemical cross-linking to VIIa after complex formation as described by Ruf et al., Proc. Natl. Acad. Sci., USA, 88:8430-8434 (1991). Antibodies to the amino acid residues at positions 94-121 of TF that encompass the inter-module junction have been shown to inhibit the binding of ligand [Ruf et al., Biochem. J., 278:729-733 (1991)], and the B.sub.C -C.sub.C -loop has been identified as a major cross-linking site in the carboxyl module of TF [Ruf et al., Proc. Natl. Acad. Sci., USA, 88:8430-8434 (1991)]. The F.sub.C -G.sub.C -loop connects two .beta.-strands that connected by disulfide bonds of two cysteine (Cys) residues in the C-module of TF. Mutational exchange of these Cys residues by Ser results in a mutant protein with diminished affinity for VIIa, consistent with a contribution of the F.sub.C -G.sub.C -loop to binding of ligand as described by Rehemtulla et al., J. Biol. Chem., 266:10294-10299 (1991).
Roy et al., J. Biol. Chem., 266:22063-22066 (1991), and Ruf et al., J. Biol. Chem., 267:6375-6382 (1991), each described TF mutants having alanine residues substituted into residue positions 165 and 166 in place of the normal human TF amino acid residues at these two positions. In these two mutants, the TF was observed to bind Factor VIIa but exhibit reduced Factor X activation, indicating that positions 165 and 166 are important for Factor X activation activity but not for ligand binding in recombinant TF proteins.
While these antibody, cross-linking and disruptive mutational analyses concordantly implicate both of the structural modules of TF in the high affinity binding of the ligand VIIa, very limited information regarding the requirements for Factor X cleavage activity is available and further the specific TF amino acid residues required for the binding of ligand have yet to be identified. The overall topographical assignment of functional and non-functional regions of TF had not been determined prior to this invention.